Purpose: SP4 is a transcription factor abundantly expressed in
retina that binds to the GC promoter region of photoreceptor signal
transduction genes. We have previously shown that SP4 may be involved in
the transcriptional activation of these genes alone or together with
other transcription factors such as SP1, neural retina leucine zipper
protein (NRL), and cone-rod homeobox gene (CRX). Since mutations in
NRL and CRX are involved in inherited retinal degenerations,
SP4 was considered a good candidate for mutation screening in
patients with this type of diseases. The purpose of this work,
therefore, was to investigate possible mutations in SP4 in a cohort
of patients affected with different forms of retinal degenerations.

Methods: 270 unrelated probands with various forms of retinal
degeneration including autosomal dominant and autosomal recessive
retinitis pigmentosa (RP), autosomal dominant and autosomal recessive
cone-rod dystrophy (CRD), and Leber's congenital amaurosis (LCA), were
screened for mutations in the SP4 gene. Single strand conformation
polymorphism (SSCP) analysis was performed on the six SP4 gene exons
including flanking regions followed by direct sequencing of SSCP
variants.

Results: Nine different sequence variants were found in 29 patients,
four in introns and five in exons. Many of the probands were previously
screened for mutations in the genes encoding the α-, β- and
γ-subunits of rod-specific cGMP phosphodiesterase (PDE6A,
PDE6B, PDE6G), the β-subunit of rod-specific transducin
(GNB1), and peripherin/rds (RDS). One group of seven probands of
Hispanic background that included five with arRP, one with RP of unknown
inheritance (isolate) and 1 with arCRD carried an Asn306Ser mutation in
SP4. Of the seven, the isolate case was homozygous and the other 6
heterozygous for the variant. Two arRP and the arCRD probands carried an
additional intronic GNB1 variant. DNA from the family members of the
arCRD proband could not be obtained, but for the other two families, all
affected members and none of the unaffected carried both the SP4
Asn306Ser allele and the GNB1 intronic variant.

Conclusions: If mutations in SP4 do cause retinal degenerative
disease, their frequency would be low. While digenic disease with the
SP4 Asn306Ser and the GNB1 intronic variant alleles has not been
established, neither has it been ruled out. This leaves open the
possibility of a cooperative involvement of SP4 and GNB1 in the
normal function of the retina.

Introduction

Transcription factors have been shown to play an important role not
only in the biology of photoreceptors and other retinal cells, but also
as sites of mutations causing degenerative disease. For example,
mutations in CRX and NRL cause various forms of progressive
retinal dystrophies [1-4]. The SP family of transcription factors
(SP1-5) is formed by a group of proteins that selectively bind to the
"GC box" in the promoter region of many genes [5-7] via three putative
zinc finger domains of the C2H2 type [8]. Based on sequence
similarities, SP1, SP3, and SP4 are more closely related to each other
than to SP2 and SP5 [6,9] and the three have similar affinity for the GC
box [10,11]. While SP1 and SP3 are ubiquitously expressed, SP4 is most
abundant in the developing nervous system, particularly in the
hippocampus [12] and retina [13], although it is also expressed in other
tissues [10,14]. Sp1 and Sp3 knockout mice all die after E10 or
at birth, respectively. Sp4 knockout mice appear to develop normally
to birth, but after birth, many pups die by P28 and those that survive
are small and have other abnormalities [12]. In the last few years, we
have studied extensively the SP4 transcription factor and have found
that it is present in all retinal layers, interacts with CRX and NRL and
activates transcription of several rod specific genes including
PDE6B and RHO, encoding the β-subunit of
cGMP-phosphodiesterase (β-PDE) and rod opsin, respectively
[13,15,16]. Because of its specific involvement in transcription of rod
genes and because of the history of transcription factor mutations
causing retinal degeneration, we considered SP4 a good candidate
gene to screen for missense mutations in patients with various forms of
retinal degeneration. To this end, we screened the 6 exons of the
SP4 gene in a group of 270 patients with various forms of retinal
degeneration that had been screened previously for a number of
photoreceptor genes [17-21]. Although we could not establish that
mutations in the SP4 gene cause retinal degenerative disease,
neither could we rule this possibility out because the families of two
patients in which an SP4 missense mutation and a GNB1 intron 2
variant were present, segregated with disease. Interestingly, the
inherited retinal degeneration of the Rd4 mouse is caused by an
inversion of mouse chromosome 4, and the site of the telomeric
breakpoint is precisely on intron 2 of the Gnb1 gene [22].

Methods

Patients

270 patient probands of mixed ethnicities (56% European, 17% Asian,
13% Black, 14% Hispanic) were screened for variants in the six exons of
the SP4 gene, including 49 with autosomal dominant retinitis
pigmentosa (adRP), 103 with autosomal recessive retinitis pigmentosa
(arRP), 26 with autosomal dominant cone-rod dystrophy (adCRD), 52 with
autosomal recessive cone-rod dystrophy (arCRD), and 40 with Leber's
congenital amaurosis (LCA). Many of the above patients had been
previously screened for mutations in the genes encoding rod- αPDE,
βPDE, γPDE, rod β-transducin and RDS-peripherin. 95
controls with a similar ethnic distribution (58% European, 16% Asian,
13% Black, 13% Hispanic) were screened for each of the above genes and
SP4 as well. Written informed consent was obtained in compliance
with the tenets of the declaration of Helsinki and with the approval of
the office of Human Research Protection of the School of Medicine,
University of California, Los Angeles.

Polymerase chain reaction

Blood was drawn in 10-20 ml aliquots and DNA was extracted from the
leukocytes by standard methods. Initial screening was done by SSCP as
described previously [17-21]. The exons of SP4 were amplified by
polymerase chain reaction (PCR) directly from genomic DNAs with
appropriate primers pairs. Each PCR amplicon included 50-150 nt of
intronic flanking sequence on each side of the exon. The PCR protocol
was 94 °C for 3 min followed by 30 cycles of 94 °C for 45 s,
55-60 °C for 45 s and 72 °C for 45 s, followed by 5 min at 72
°C. The sequences of primer pairs are presented in Table 1.

Single strand conformation polymorphism

Amplicons were separated by electrophoresis in 7% acrylamide gels and
analyzed by standard P32 autoradiography or silver staining
methods to reveal polymorphisms as described previously [17-21].

SSCP screening of the SP4 gene showed 9 sequence variants in 29
patients, five present in exons (Table 2). The heterozygous Leu241Val
and Pro286Ala missense variants, both present in exon 3 of arRP probands
did not segregate with disease in the corresponding families. An
Asn306Ser missense variant in exon 3 was present in both alleles of one
isolate RP proband, in one allele of five arRP probands and in one arCRD
proband. This missense variant was also present in 1/95 controls.
Interestingly, although only 14% of the 270 patients and 13% of the 95
controls were Hispanic, all seven patients and the 1 control that
carried Asn306Ser were Hispanic. Thus, 18.4% (7/38) of the Hispanic
patients carried Asn306Ser while none of the other patients did,
including 0/151 patients of European origin. The other 2 coding region
variants were both silent. Neither was present in 95 controls. The
remaining 4 intronic sequence variants were present in patients and
absent from controls with the exception of -121 A to C which was present
in one control (Table 2).

Table 3 shows the results of previous screenings of several
photoreceptor genes in the six probands with the Asn306Ser mutation in
one allele. Patient 856 has arCRD while the other 5 patients have arRP.
Three of the six probands also carried an A-G variant in intron 2 of the
GNB1 gene. DNAs of the family members of one of these probands could
not be obtained (family 2177). However, in the families of the other two
probands, the SP4 missense and the GNB1 intronic variant
segregated with disease (Figure 1A,B). None of the other variants in
the screened genes segregated with disease. We found no additional
variants in the genes encoding α-, β- and
γ-cGMP-phosphodiesterase, RDS/peripherin or the β-subunit of
transducin in the RP isolate patient homozygous for Asn306Ser.

Discussion

Even though 2/3 of mice born with the Sp4 gene deleted die within
the first four weeks of life [14], the surviving mice have many
abnormalities including severe retinal degeneration (our data, not
shown). Therefore, we considered the human SP4 gene a good candidate
for the site of missense mutations causing retinal degeneration, given
its involvement in the transcription of several photoreceptor genes
including PDE6B [13,15,16] and the phenotype of the Sp4 knockout
mouse.

We found nine unique sequence variants in the SP4 gene of 29
patients affected with different types of inherited retinal degenerative
disease. Of these, five variants were in one of the six exons of
SP4. Two of the variants coded for the same amino acid (Ala276Ala
and Gln451Gln). Two more variants predicted amino acid changes in the
SP4 protein, Leu241Val and Pro286Ala, but neither segregated with
disease in the corresponding families. The fifth missense Asn306Ser
mutation was present in seven probands. One of the probands that had the
homozygous Asn306Ser mutation was an isolate with RP, so we could not
tell if the two Asn306Ser alleles were causing disease. In family 449,
an affected sibling of the proband did not carry the Asn306Ser allele;
for the probands of families 856 (arCRD) and 1824, neither a second
variant SP4 allele nor a variant in any of the other genes
previously screened could be identified. The three remaining probands
all carried Asn306Ser and an intronic A-G substitution 103 bp upstream
of the 3' splice site of intron 2 of the GNB1 gene. We could not
obtain DNAs from the family of one of the probands (2177), but in the
families of the other two probands (485 and 1526) only the two affecteds
in each pedigree carried both alleles (Figure 1A,B). Both of these
families had arRP. All seven families carrying Asn306Ser were of
Hispanic background and so was the 1 control carrying the same mutation.
Thus, 18.4% of the Hispanic patients carried this allele while none of
the patients from other backgrounds did (0/151 Europeans, 0/35 Blacks
and 0/46 Asians). The higher frequency of Asn306Ser in Hispanic patients
(p<0.001 applying the Fisher's exact test) compared to a relatively
low frequency in Hispanic controls (1/12=8.3%) suggests that this
variant may be pathogenic.

There are several additional reasons that implicate SP4 Asn306Ser
as a mutant allele that may contribute to autosomal recessive disease.
(1) We have no family history for the proband that carried two alleles
of Asn306Ser. Therefore, the two Asn306Ser alleles together could be the
cause of that isolate patient's disease. (2) In each of the three
families where the Asn306Ser allele did not segregate with disease, two
mutant alleles in other genes may have rendered the presence of the
heterozygous Asn306Ser coincidental and unrelated to disease. However,
this does not rule out the possibility that two Asn306Ser alleles could
cause disease. (3) For the two families where only the affecteds carried
both the Asn306Ser allele and the GNB1 variant intronic allele,
pathogenesis is possible at least genetically. Furthermore, GNB1 has
three GC boxes in its promoter and SP4 interacts with GC boxes. Thus,
digenic disease may be plausible. To answer the question of
pathogenicity of Asn306Ser, functional assays of the protein carrying
this variant would have to be conducted. Nevertheless, the possibility
that Asn306Ser may be pathogenic is supported by the fact that Asn306Ser
is in the transactivation domain of the SP4 protein and in one of six
glycosylation sites (N-X-S/T; Figure 2). Changing asparagine to serine
eliminates this site of posttranslational modification and this may
affect the function of the protein. Interestingly, at position 306 there
is an asparagine only in the human SP4 sequence while in the mouse, rat,
dog and cow there is a threonine. Therefore, asparagine is not a
conserved residue. With regard to the GNB1 intronic variant, it is
not in a splice site or a consensus branch point, but it may be in a
heretofore-unknown regulatory region.

For the intronic GNB1 variant, a DNA fragment including exon 2,
intron 2 (carrying the variant), and exon 3 would have to be expressed
to determine if this variant caused a splicing problem. However, the
sequences adjacent to the A-G substitution do not correspond to
consensus branch point sequences. Another possibility is that the A-G
substitution would disrupt an enhancer or a repressor sequence causing
altered expression of the GNB1 gene. Although there is no direct
evidence that the Asn306Ser mutation in the SP4 gene and the intron
2 A-G variant of the GNB1gene together are responsible for disease
in the affected individuals, digenic disease cannot be ruled out without
further testing the pathogenicity of the alleles. It is certainly
plausible that the protein products of a phototransduction gene like
GNB1 and a transcription factor that may influence its expression
like SP4 can together cause digenic disease when one allele of each
carries a mutation.

Acknowledgements

This work was done with the support of a grant from the Foundation
Fighting Blindness and grant EY02651 from NIH to DBF.

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